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United States Patent 0 PROCESS FOR PRODUCING A HIGH PROTEIN COMPOSITIONBY CULTIVATING MICRO- ORGANISMS ON AN N-ALIPHATIC HYDRO- CARBON FEEDJohn D. Douros, Jr., Littleton, Colo., assignor to Esso Research andEngineering Company, a corporation of Delaware No Drawing. Original No.3,308,035, dated Mar. 7, 1967, Ser. No. 410,299, Nov. 10, 1964.Application for reissue Apr. 4, 1967, Ser. No. 637,018

11 Claims. (Cl. 195-28) Matter enclosed in heavy brackets appears in theoriginal patent but forms no part of this reissue specification; matterprinted in italics indicates the additions made by reissue.

ABSTRACT OF THE DISCLOSURE Fermentation process for producing a highprotein composition on an aliphatic hydrocarbon feed in a mediacomprising aqueous growth medium containing oxygen and other essentialcell nutrients.

This invention is directed to a method for biosynthesis Reissued Dec.10, 1968 of any of eight certain microorganisms having an unusual-Microorganism name:

A.T.C.C. number Pseudomonas ligustri 15522 Pseudomonas pseudomallei15523 Pseudomonas orvilla 15524 Alcaligenes sp. 15525 Cellumonas galba15526 Brevibacterium inspectiphilium 15528 90 Corynebacterium sp. 15529Corynebacterium pourometabolum 15530 The bacteriological characteristicsof these microorganisms as determined by the below indicated testsleading NOMENCLATURE TESTS 25 to the above nomenclature are as follows:

Tests, A.T.0.0. N0.

Morphology Small gram negative rod Smal, thin gram negative Swag, thingram negative Small gram negative rod.

r0 to Motility (Motile) (Immotile). Gram Reaction (Negative) Agar ColonyMorphology Opalescent, filamentous, Raised entire Raised, rough,circular, Rough, circular, si. eleradiated surface, ridged, suriace,glistemn undulate opaque, voted edge unduiate,

rhizoid butyrous.

viscid. on glucose only opaque, membranous.

Carhonhydratc Fermentation on starch and glucose--- on glucose only onglucose, on starch,

lactose, sucrose, mannitol.

Pigmcntation Green on tryptose, brown White on gotato, nutrient White onall media fili- White on potato green on on nutrient agar, white agar antryptose, on form. tryptosc.

on potato, echinnlate. dextrose (produces green pigment] GelatinLiquefaction Growth Temperature C.)- 30 (37, 42) 30 (37). Growth p11 7.54.0-9. Urea Hydrolysis- Sulfide Production. Cataiase Production...Nitrate Reducticn., Oxygen Aerobe. Aerobc. Source Soil Soil son. HabitatSoil-hydrocarbons Soil-hydrocarbons SoiLhydrocarbons.

NOMENCLATURE TESTS Tests, A.T.C.C. No.

Morphology Small thin gram positive Small gram positive rod Long rodsome snapping. Pleomorphlc gram positive rod. rod, some bending.Motility (lrnmotile) Gram Reaction (Positive) 4- (Positive) 1 AgarColony Morphology Lobate, fiat, smooth Lightish green raised Raisedbutyrous opaque- Convey entire edge opaque, membranous. smooth opaque.creamy.

Carhonhydrate Fermentation on glucose, lactose,

sucrose, starch and mannitol.

Pigmentation White on nutrient agar,

yellow tryptone, white on potato.

Gelatin Liquefaction Growth Temperature C 0.).

Catalase Production Nitrate Reduction- I Soil-hydrocarbons (Positive) onglucose, on

lactose, sucrose, starch and mannitoi.

White on dextrose,

greenish on tryptone.

(Negative).

(None fermented) Cream on potato dextrose trypt/one.

White on dextrose, cream on potato and tryptonc.

Aerobe.

Each of these eight microorganisms has a valuable composite ofproperties, viz., a high protein content in excess of 50 percent, anessential amino acid index in excess of 45 and an excellent amino acidprofile, as will be more specifically indicated hereinafter. Moreover,said microorganisms are non-toxic and thus can be used in animal feedsupplements. The protein can be extracted from these microorganisms byconventional extraction procedures and the protein extract then used asa glue or adhesive. A suitable protein extraction procedure involvessequential lysing, e.g., with acetone or other suitable organic lysingsolvent, basic or acid extraction and isoelectric precipitation.Intracellular and/or extracellular amino acids can be isolated from themicroorganism cells and/or fermentation media. In this regard theprocess of the present invention can be visualized as a combined processfor biosynthesis of cells and chemicals.

The process of this invention is conducted by cultivating (fermenting)any of the aforesaid microorganisms on an aliphatic hydrocarbon feedsource, e.g. a C -C n-paraffin feed in an aqueous growth mediumcontaining available oxygen and other essential cell nutrients for saidmicroorganisms thereby producing and accumulating said microorganisms,and thereafter isolating said microorganism cells. If these cells are toform part of an animal feed supplement, the cells are usually renderednonviable before use.

The present world shortage of protein is well known. In an attempt toalleviate this protein shortage recently there have been developedbiosynthesis procedures whereby protein can be provided by the growth ofbacteria on various carbon-containing substrate materials. One knowntechnique of protein biosynthesis involves growing yeast on carbohydratesubstrates. However, most of these biosyntheses require expensivevitamins and/or other growth mediums in addition to the comparativelyexpensive carbohydrate feeds in order to attain the desired microbialcell growth.

Another recent technique for biologically synthesizing protein, but invery small yield is the culture of microorganisms on petroleumsubs.rates to produce esters and chemicals as a major product andmicrobe cells as a byproduct in very small amounts. This latter type ofprotein synthesis usually involves the use of less expensivecarbon-containing feed materials, e.g., hydrocarbons rather thancarbohydrates; but has not attained wide acceptance due to thedifficulty of securing microbe cells having a high protein contentcoupled with a sufliciently high essential amino acid index. Otherproblems frequently connected with biosyntheses using hydrocarbon feedstocks are low cell growth rates (extremely long residence times) andinability of the microbe cells to effectively uiilize hydrocarbon feedsfor growth and reproduction.

The process of the present invention constitutes a marked improvement inprotein biosynthesis by securing productive growth of the aforesaidmicroorganisms having a valuable combination of properties, in goodyield at attractive growth rates, while using comparatively inexpensiveC -C aliphatic hydrocarbon feeds, e.g., C C n-parafiins, C C olefins,etc., for microbial growth. Moreover, the microorganisms contemplatedherein can be isolated readily from the biosynthesis bath in which theyare grown thus further enhancing the economic merits of the presentinvention.

For the culture medium in which the microbiological cells having theabove-mentioned high protein content and high essential amino acid indexare reproduced and accumulated in accordance with this invention, C to Cn-aliphatic hydrocarbons can be used as the chief source of carbon andhydrogen for cell growth. Usually, however, the source of carbon will beC to C n-parafins, e.g., C to C light naphthas (viz., low boilinghydrocarbon oils of the C H series and having a boiling point between 95and about 150 C.) and petroleum fractions containing them, and C to Cgas oils boiling in the range of about 190 to 320 C., and petroleumfractions containing them. The preferred n-paraffin feed for themicrobes contemplated herein are the C to C n-paratfins. Each of theabove feeds can and frequently does contain normal olefins, e.g., C -Cmono and polyolefins, in varying amounts, e.g., from 0.05 to 30.0percent by weight (based on total hydrocarbons in the feed).

Polycyclic aromatic compounds are usually excluded from the feed as suchmaterials are considered to be possibly carcinogenic and couldcontaminate the harvested cells in feeds.

While the presence of branched aliphatic hydrocarbons (including botholefins and alkancs) in concentrations up to 30 wt. percent can betolerated in the hydrocarbon feed; concentrations in excess of 10 wt.percent of non-normal aliphatic hydrocarbons are usually avoided becausethe aforesaid microorganisms are sclective preferentially to normalaliphatic hydrocarbons, especially C C n-aliphatic hydrocarbons.

Oxygen can be supplied to the cultivation medium in any form capable ofbeing assimilated readily by the inoculant microorganism, andoxygen-containing compounds can be used as long as they do not adverselyaffact microorganism cell growth and conversion of hydrocarbon feed tomicroorganism cells. Conveniently, how ever, the oxygen is supplied asan oxygencontaining gas, e.g., air, which contains from 19 to 22 wt.percent oxygen. While it is preferable to employ air, oxygen enrichedair having more than 22 wt. percent oxygen, e.g., enriched air having inexcess of 22 Wt. percent oxygen, can be used.

Nitrogen is essential to biosynthesis. The source of nitrogen can be anyorganic or inorganic nitrogen-containing compound which is capable ofreleasing nitrogen in a form suitable for metabolic utilization by themicroorganis m(s) being harvested. In the organic category, thefollowing compounds can be listed as exemplary nitrogen-containingcompounds which can be used; proteins, acid-hydrolyzed proteins,enzyme-digested proteins, amino acids, yeast extract, asparagine, urea,etc., which materials are utilized as carbon sources also. For reasonsof economy, it is usually preferable to employ inorganic nitrogencompounds, such as: ammonia, ammonium hydroxide, or salts thereof, suchas ammonium citrate, ammonium sulfate, ammonium phosphate, ammonium acidphosphate, etc. A very convenient and satisfactory method of supplyingnitrogen is to employ ammonium phosphate or ammonium acid phosphate,which can be added as the salt, per se, or can be produced in situ inthe aqueous fermentation media by bubbling nascent nitrogen through thebroth to which phosphoric acid was previously added, thereby formingammonium acid phosphate.

In addition to the energy and nitrogen sources, it is necessary tosupply requisite amounts of selected mineral nutrients in the feedmedium in order to insure proper microorganism growth and maximizeselectivity, viz., the conversion of hydrocarbons to microorganismcells. Thus, potassium, sodium, iron, magnesium, calcium, manganese,phosphorous, and other nutrients are included in the aqueous growthmedium. These necessary materials can be supplied in form of theirsalts, and preferably their water-soluble salts. For example, thepotassium can be supplied as potassium chloride, phosphate, sulfate,citrate, acetate, nitrate, etc. Iron and phosphorus can be supplied inthe form of sulfates and phosphates, respectively, e.g., iron sulfate,iron phosphate. Usually most of the phosphorus is supplied as ammoniaphosphates. When either ammonium phosphate or ammonium acid phosphate isused, it can serve as a combined source of both nitrogen and phosphorus(phosphate ion) for microorganism cell growth. Generally thecompositional content of the fermentation growth media at the outset offermentation is as follows:

Other optional mineral nutrients which can be included in trace amountsinclude:

Of course, the essential and optional nutrients can be supplied in theform of other salts than those tabulated hereinabove.

The temperature of the culture during biosynthesis can be varied fromabout to about 55 C. depending upon the specific microorganism beinggrown, but usually temperatures of from about 20 to 45 C. are employed.Preferably the fermentation is conducted at temperatures ranging fromabout to C. According to the present invention cultivation is conductedin a medium as described above by adding an inoculum of themicroorganism to be harvested to the fermentation media containing then-aliphatic hydrocarbon feed source and regulating the pH usually fromabout 6 to about 8 while maintaining proper growth temperatures undershaking or stirring while utilizing aeration in submerged condition. Ifthe pH becomes too high for optimum growth of the microorganism to beharvested, it can be lowered readily to addition of a suitable acid tothe fermentation media, e.g., HCl. In like manner if the pH becomes toolow, it can be raised by addition of a suitable base, e.g., ammonia orammonium hydroxide.

At the start-up of the fermentation the growth medium is inoculated withthe microorganism to be harvested, e.g., by use of a previouslycultivated inoculum in the same media in which it is to be grown, e.g.,as described above. The initial concentration of inoculum containingsaid microorganism at the outset of fermentation can vary widely, e.g.,0.0005 to 50.0 grams per liter of total fermentation media. Otherinoculation procedures can be employed, e.g., use of an inoculum wheresaid microorganism is previously grown on a media different from that inwhich the fermentation is to be conducted and then transferred to thefermentation vessel(s) etc.

At the end of fermentation, the cells are isolated from the fermentationmedia by decantation, filtration (with or without filter aids),centrifugation, etc.

The filtered cells can then be dewatered, e.g., using rotary drumdryers, spray dryers, etc, although this is not absolutely necessary.The cells are usually rendered non-viable before use by spray drying at150185 C. for from 230 seconds. Care should be exercised duringpasteurization to avoid extreme temperatures for extended time periodswhen the harvested cells are to be used as protein supplement (in orderto avoid protein degradation).

If the cells are to be used in making glues, adhesives, etc., it is notnecessary to render them non-viable as the protein extraction proceduressuffice. The same is true when the microorganism cells are grown andharvested for their intracellular chemicals, e.g., amino acid, content.

The present invention will be illustrated in greater detail by theexamples which follow, but these examples should not be construed aslimiting the scope thereof.

EXAMPLE 1 A growth medium of the following composition was prepared:

Concentration Component: (grams liter) n-Hexadecane 20.0 K HPO s 5.0(NH4)3HP04 10.0 N21 SO MgSO,-7H O 0.4 FeSO 'H O 0.02 MHSO4'4H2O NaCl0.02

Water (sufficient to make a volume of 100 mls.).

Commercial n-hexadecane containing 1 wt. percent C10 nmonoolefin.

After regulating the pH to 7.2 to 7.7, the above media was introducedinto a 500 ml. Erlenmeyer flask, and the flask contents were sterilizedby heating at 121 C. for 15 minutes. Then approximately 0.001 gram perliter of Brevibacterium insectiphilium (A.T.C.C. No. 15528), previouslycultured for 48 hours at 30 C. on the same medium, was inoculated intothe fermentation growth medium. The fermentation media was culturedunder shaking at 30 C. for 48 hours maintaining the growth pH between 6and 7.5 throughout fermentation.

After 48 hours, the cell concentration was 6.2 grams per liter, and 40wt. percent of the n-hexadecane aliphatic hydrocarbon feed was utilizedby the microorganism (cell yield of 77.5 percent based on aliphatichydrocarbon utilized). After the completion of fermentation, the brothwas centrifuged and then sterilized in a spray drier at 180 C. for 4seconds.

EXAMPLE 2 Corynebacterium sp. (A.T.C.C. No. 15529) was fermented for 48hours at 30 C. using the same growth medium and procedure set forth inExample 1 with the exception that 10 grams per liter of n-hexadecane wasused in this fermentation. After 48 hours, the cell growth was 9.5 gramsper liter and 100 percent of the n-hexadecane hydrocarbon feed wasutilized (cell yield of percent based on aliphatic hydrocarbonutilized).

EXAMPLE 3 Corynebacterium pounometabolum (A.T.C.C. No. 15530) was grownfor 48 hours at 30 C. at a pH of 6 to 7 in accordance with the procedureof Example 1 and using the growth medium thereof, but with ann-hexadecane concentration of 17 grams per liter. After 48 hours thecell growth was 12.8 grams per liter and percent of the n-aliphatichydrocarbon feed was utilized (cell yield of 75 percent based onn-hexadecane utilized).

EXAMPLE 4 Pseudomonas ligustri (A.T.C.C. No. 15522) was grown for 48hours at 30 C. at a pH of approximately 7.8 under the procedure ofExample 1 only using 17 grams per liter of n-hexadecane. After 48 hours,the cell growth was 9 grams per liter and 99.9 percent of then-hexadecane was utilized by the microorganism (cell yield ofapproximately 51 percent based on utilized n-aliphatic hydrocarbonfeed).

EXAMPLE 5 Pseudomonas pseudomallei (A.T.C.C. No. 15523) was grown for 48hours at 30 C. at a pH range of 7 to 8 as in Wt. percent of Amino Acidin Harvested Cells from Exzunple Nu1nber TABLE 2 (AMINO ACID PROFILE)Essential Amino Acid Example 1, but using 17 grams per liter ofn-hexadecane. After 48 hours, the cell growth was 6 grams per liter and52 percent of the n-hexadecane was utilized (cell yield of 70.7 percentbased on n-aliphatic hydrocarbon feed utilized).

Moreover, this invention also has a chemicals aspect in serving as abiosynthetic synthesis of intracellular and extracellular chemicals. Inthe latter respect it should be noted here that further experimentalfermentation biosynthesis using the microorganisms of Example 7(A.T.C.C. No. 15525) and Example 8 (A.T.C.C. No. 15526) respectively onfermentation growth mediums having 4-16% concentrations of n-aliphatichydrocarbon (n-hexadecane) feed produced 1.109 grams per liter and 0.326gram per liter, respectively, of extracellular amino acid mixtures.These fermentations were conducted at 30 C. under shaking for 72-144hours in two stages with the first stage constituting a 24-48 hourgrowth on naliphatic hydrocarbon (to cell growth levels ranging from 0.8to 9.4 grams per liter) followed by a 24-96 hour second stage at thesame conditions in which varying amounts, viz., 6-10% of an n-alkylatedbenzene (n-amyl benzene) were added. The harvest of cells plus aminoacids was performed subsequent to the addition of the n-alkylatedbenzene(s) or mixtures thereof.

While the above examples involve 48-hour fermentations, the fermentationperiod can be varied widely from about 30 minutes to continuousoperation. Usually in batch fermentations to maximize cell yield andmaintain an economically advantageous cell growth level, fermentationwill be conducted for time periods ranging from 1 to days and morepreferably from 36 to 96 hours.

While the preceding examples illustrate the present invention in greatdetail, it should be remembered that the present invention in itsbroadest aspects is not necessarily limited to the specific materialsand conditions shown in these examples.

What is claimed is:

1. A process for biosynthetically producing a high protein compositionhaving a protein content in excess of 50 percent and an essential aminoacid index in excess of 45 which comprises cultivating a microorganismselected from the group consisting of:

Pseudornonas ligustri (A.T.C.C. No. 15522), Pseudomonas pseudomallei(A.T.C.C. No. 15523), Pseudomonas orvilla (A.T.C.C. No. 15524),Alcaligenes sp. (A.T.C.C. No. 15525), Cellumonas galba (A.T.C.C. No.15526), Brevibacterium insectiphilium (A.T.C.C. No. 15528),Corynebacterium sp. (A.T.C.C. No. 15529), and Corynebacteriumpourometabolum (A.T.C.C. No.

on an n-aliphatic hydrocarbon feed in a media comprising an aqueousgrowth medium containing oxygen and other essential cell nutrients attemperatures ranging from about to 55 C., and harvesting by centrifugingand spray drying said microorganism cells.

2. A process according to claim 1 wherein said n-aliphatic hydrocarbonfeed is a C -C n-aliphatic hydrocarbon feed.

3. A process according to claim 1 wherein the concentration of saidn-aliphatic hydrogen feed ranges from 4 to 120 grams per liter.

4. A process according to claim 1 wherein said cultivation is conductedbatchwise for time periods ranging from about 24 to 120 hours.

5. A process according to claim 1 which includes heating said harvestedcells at temperatures ranging from about 150 to about 185 C. to renderthem non-viable.

6. A process according to claim 1 wherein said aqueous growth mediumincludes the following components in the below tabulated concentrations-Concentration Component: gram/ liter n-Aliphatic hydrocarbon 4-120 KHPO. 0.5-15 (NH HPO 5-15 Na,.so, 0.1-10 FeSO -7H O 0002-005 MgSO 7H O0.1-0.7 MnSO -4H O 0002-005 NaCl 0.002-0.05

7. A process according to claim 1 wherein said n-aliphatic hydrocarbonfeed is predominantly a (D -C nparaffin.

8. A process for biosynthetically producing a high protein compositionhaving a protein content in excess of 50 percent and an essential aminoacid index in excess of 45 which comprises cultivating a microorganismselected from the group consisting 0 Pseudomonas ligustri (A.T.C.C. No.15522), Pseudomonas pseudonwllei (A.T.C.C. No. 15523), Pseudomonasorvilla (A.T.C.C. No. 15524), Alcaligenes sp. (A.T.C.C. 15525),Cellumonas galbat (A.T.C.C. No. 15526), Brevibactierium insectiphilium(A.T.C.C. No. 15528), Corynebacteritrm sp. (A.T.C.C. No. 15529), andCom'nebacteriurn pouro'metabolum (A.T.C.C. No.

Concentration Component: (gram liter) n-Aliphatic hydrocarbon 4-120 KHP0 0.5-15 (NH HPO 5-15 Nd SO FeSO -7H O 0.002-0.05 M s0,-7H 0 0.1 0.7MnSO -4H O 0002-005 NaCl 0002-005 patent.

UNITED STATES PATENTS 4/1963 Rudy et al -80 12/1965 Iizuka et al 195-29LIONEL M. SHAPIRO, Primary Examiner.

U.S. Cl. X.R.

